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Fig. 4 | Microbial Cell Factories

Fig. 4

From: The TetR-type regulator AtsR is involved in multidrug response in Corynebacterium glutamicum

Fig. 4

AtsR bound directly to the promoter region of the ncgl0887-atsR operon. A, B EMSA was performed to analyze the interaction between AtsR and the promoter DNA fragment of the ncgl0887-atsR operon (Pncgl0887) or the ncgl0884 promoter DNA fragment (Pncgl0884). 232-bp and 205-bp DNA fragments amplified from the ncgl0887 coding region using the primers control F1 and control R1 instead of the 232-bp Pncgl0887 (control A, lane 7) and the ncgl0884 coding region using the primers control F2 and control R2 instead of the 205-bp Pncgl0884 (control A, lane 7), respectively, and an irrelevant protein BSA instead of AtsR (control B, lane 8) in the binding assays were used as negative controls to determine the binding specificity of AtsR. C Identification of the AtsR-binding sites within the Pncgl0887. Localization of the AtsR binding site using the ncgl0887-atsR operon promoter fragments (designated A–G) and purified AtsR in EMSAs. The numbers showed the position of the fragments relative to the translational start codon of the target gene. ‘+’and ‘−’ signs indicated whether the fragment was shifted by AtsR. D The interaction between AtsR and the promoter DNA fragment of the ncgl0887-atsR operon mutating the identified AtsR binding regions (Pncgl0887M). E Mutations in the identified AtsR binding site derepressed the expression of the ncgl0887-atsR operon. n.s., not significant. F The 12-bp proposed AtsR consensus binding site (bold). The mutations M1–M3 were introduced by PCR and were shown below the wild-type (WT) sequence. The corresponding DNA fragments were analyzed by EMSAs with AtsR

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